USE OF HYALURONIC ACID DERIVATIVES IN THE REGENERATION OF BONE AND CARTILAGE TISSUE

Abstract

The present invention relates to the use of derivatives of hyaluronic acid, heterocyclic compounds and amino acids of natural origin in individual, oligomeric or polymeric form for the treatment of skeletal diseases, particularly in the regeneration of bone and cartilage tissues.

Description

USE OF HYALURONIC ACID DERIVATIVES IN THE REGENERATION OF BONE AND CARTILAGE TISSUE FIELD OF INVENTION The present invention relates to the use of derivatives of hyaluronic acid, heterocyclic compounds and naturally occurring amino acids in individual, oligomeric or polymeric form for the treatment of skeletal diseases, particularly in the regeneration of bone and cartilage tissues. BACKGROUND OF THE INVENTION In the biological field, a tissue is defined as a group of structurally similar cells associated by function. Therefore, it constitutes a higher level of cellular organization, with a specific role to play within an organism. With regard to the animal kingdom, including humans, four fundamental tissue types can be recognized: epithelial, connective, muscular, and nervous, each further divided into more specialized subtypes. In higher animals, different tissues combine to form additional organized structures: organs. Connective tissues, such as bone, adipose, fibrous, and trophic tissues, are tissues consisting of separate cells separated by a non-living material called the extracellular matrix (ECM). This matrix can be liquid or rigid; two extreme examples are blood, in which the matrix is ​​plasma, and bones, which have mineralized tissue and an extremely rigid matrix. It is precisely because of this peculiar characteristic that bone tissue is sometimes referred to as hard tissue, in contrast to soft tissues, which generally refers to the other connective tissues. Cartilage, the precursor tissue of bone, is also part of the connective tissues. It is composed mainly of cells called chondrocytes, which are capable of producing a high amount of extracellular matrix composed primarily of collagen, elastin, and proteoglycans. Tissue regeneration, which must take place after injuries or illnesses in order to ensure complete healing, is a process based on the renewal and differentiation of the cells of the tissues involved. Regenerative medicine is an emerging field of research that has gained significant interest in recent years. It combines various aspects of medicine, cell biology, and bioengineering with the ultimate goal of regenerating, repairing, or replacing damaged or missing tissues. Different lines of research, involving both differentiated cells and stem cells, aim to optimize the regeneration, healing, and / or replacement of damaged tissues. One of the most widely used approaches in the field of bone and cartilage tissue regeneration today is based on a mesenchymal stem cell (MSC) approach. Regarding cartilage, although cartilage differentiation from MSCs has been demonstrated, the clinical use of this approach is still limited and not entirely successful. Cartilage regeneration can also be achieved through methods such as tissue grafting (autografts or allografts) or through techniques described in the literature adapted to stimulate the natural repair process. The techniques identified to date as the most reliable for cartilage tissues, therefore, aim to enhance the regenerative properties of the tissues or involve chondrocyte transplantation to augment or heal residual tissue. Regarding bone tissue, bone marrow remains the source of choice for isolating MSCs from which to obtain differentiated bone cells, but other sources for obtaining MSCs, such as dental tissue, are also known. Finally, the extracellular matrix is ​​known to play an important role in cell differentiation in connective tissues; in particular, the interaction of MSCs with the ECM can enhance the osteogenic differentiation of these cells. In fact, the ECM contains a variety of macromolecules, including collagen, adhesive glycoproteins, and glycosaminoglycans (GAGs), which not only support cells and determine tissue structure but also contribute to the propagation of growth factors and cell interactions with the microenvironment, thus influencing cell behavior. Despite the numerous studies and progress made in recent years in the context of the use of stem cells, the reconstruction of bone and cartilage tissue defects remains a challenge for regenerative medicine, as current known treatments are only partially effective and not always feasible. Therefore, there is a need to find new ways and approaches to more easily and effectively address the problem of regeneration and healing of the body's bone and cartilage tissues. BRIEF DESCRIPTION OF THE INVENTION A class of hyaluronic acid derivatives, in which this molecule is associated with at least one heterocyclic compound derived from a purine or rhimidine base and with at least one other organic compound consisting of a naturally occurring amino acid, in individual, oligomeric or polymeric form, is described in EP1525244 with procedures for its preparation. ML / t / ZUZZ / U / Ί The aforementioned hyaluronic acid derivatives, a glycosaminoglycan (GAG) naturally present in the extracellular matrix (ECM), are more stable than the native form because the compounds associated with them are located at the target sites of the lytic enzyme hyaluronidase, normally responsible for its degradation, thus hindering its action. Furthermore, depending on the type of heterocyclic compounds and amino acids selected, these derivatives exhibit a particular three-dimensional structure, enabling them to modify the ECM microenvironment. Therefore, the object of the present invention is the use of hyaluronic acid derivatives in the regeneration of bone and cartilage tissues, thanks to the high regeneration potential in tissues highlighted in the experimental section accompanying this description. Hyaluronic acid compounds and at least one purine and / or pyrimidine-derived heterocycle, associated with at least one different organic compound selected from naturally occurring amino acids in individual, oligomeric, or polymeric form, are used, according to the present invention, in the treatment of skeletal conditions, particularly in the regeneration of hard tissues. According to the present invention, the hyaluronic acid derivatives whose use is the subject of the present invention are high molecular weight hyaluronic acid, in the range between 400,000 and 4 million Da, preferably between 800,000 and 3.5 million Da, more preferably between 1.5 and 3 million Da. Optionally, the hyaluronic acid derivatives whose use is the subject of the present invention consist of low molecular weight hyaluronic acid, for example, in the range between 80,000 and 400,000 Da. The molecular weights of polymers in general, and of hyaluronic acid in particular, are, for example, the number average molecular weight Mn, defined as the average of the weights of the polymer chains: Mn = Z(¡)N¡M¡ / Z(¡)N¡ where M¡ is the molecular weight and N¡ is the number of chains or the average molecular weight Mw which is defined as: Mw= Z(¡) N¡M¡2 / Z(¡)N¡M¡ This amount is more influenced by the fraction with higher molecular weights and is higher than the average molecular weight. Determining the average molecular weight of a polymer is of great importance, as it represents the polymer's main characteristic to which many of its properties are related. Molecular weight can be obtained using various techniques, including centrifugation (sedimentation equilibrium), light scattering, and ΜΛ / t / ZUZZ / U / Ί Z40 osmometry. The rate at which molecules settle in an ultracentrifuge is proportional to their molecular weight: assuming, in fact, that the molecular weight increases as its volume increases, the molecular weight can be determined as a function of the sedimentation rate. The technique of light scattering is based on the principle that when a beam of light travels through empty space in a straight line, it loses no energy along the way. However, if particles of any kind are present in the space, the light beam will be scattered or deflected in all directions by these particles. The primary light beam loses some of its energy and its intensity decreases. It is possible to develop a theory for the determination of the molecular mass of a polymer M by measuring the intensity of the scattered light and, therefore, the photodiffusion of the polymer itself placed in a dilute solution in a suitable solvent. The molecular mass obtained has an average value and it can be shown that in this case it is the average by weight. Of the methods mentioned above, the most important and widespread is osmometry. The osmotic pressure (π) is measured for solutions with different concentrations of polymer C. Recall that π = CRT When the temperature T at which the measurement is carried out is known, the value of the molecular weight will be calculated, remembering that C= mass / molecular weight. The most commonly used standard technique for determining the chemical-physical characteristics of a polymer is called GPC (Gel Permeation Chromatography). According to the present invention, the selected heterocyclic compounds are derivatives of selected purine bases, for example, adenine and guanine, and / or selected pyrimidine compounds, for example, thymine, cytosine, and uracil. The preferred base, according to the invention, is a pyrimidine base such as thymine. Other purine or pyrimidine derivatives that can be used to form compounds whose use is the subject of the present invention can be selected from: 5,6-dihydrouracil, 1-methyluracil, 3-methyluracil, 5-hydroxymethyluracil, 2-thiouracil, N4-acetylcytosine, 3-methylcytosine, 5-methylcytosine, 5-hydroxymethylcytosine, 1-methyladenine, 2-methyladenine, 7-methyladenine, N6-methyladenine, N6,N6-dimethyladenine, N6-(A2-isopentenyl)adenine, 1-methylguanine, 7-methylguanine, N2-methylguanine, N2,N2-dimethylguanine. Preferably, in the hyaluronic acid derivatives whose use is the subject of the present invention, the interaction between the hyaluronic acid chain and the purine or pyrimidine bases takes place thanks to at least one ionic bond between a -COOH residue of the acid and ML / t / ZUZZ / U / 1Z40 a basic center, in particular a basic nitrogen, of heterocyclic bases. In practice, according to the present invention, hyaluronic acid is reacted with at least one purine and / or pyrimidine base selected from those established above, under reaction conditions that allow the formation of at least one type of ionic bond between at least one acid center of the hyaluronic acid, such as, for example, a free carboxyl group in the form of an acid salt or carboxylate, and at least one basic center of the purine and / or pyrimidine base, also in the form of a free base or ammonium salt. According to the present invention, the hyaluronic acid derivatives whose use is the subject of the present invention may contain more than one type of purine and / or pyrimidine base with a variable reciprocal ratio; the derivatives may, therefore, be represented by mixed salts consisting of a variable amount of purine / pyrimidine bases. The hyaluronic acid derivatives, the use of which is the subject of the present invention, also include at least one naturally occurring amino acid, or an oligomer or polymer thereof, to provide an additional salting product thanks to the -COOH groups present and remaining free in the hyaluronic acid structure. The amino acids that can be used to form these derivatives are selected, for example, from: alanine, arginine, asparagine, aspartic acid, glutamic acid, cysteine, phenylalanine, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, proline, serine, tyrosine, threonine, tryptophan, and valine. Preferably, the amino acids are selected from lysine and alanine. The characteristics of the derivatives reflect those of the hyaluronic acid, purine and / or pyrimidine bases and amino acids, which in turn are linked by at least one easily hydrolyzable ionic bond, making the different components readily accessible in situ. The compound currently marketed under the name TLysYal® (T-Lys), a derivative of hyaluronic acid, lysine and thymine, is particularly preferred due to the formation of ionic-type bonds. According to the present invention, compounds consisting of hyaluronic acid, at least one heterocyclic compound selected from a purine and / or pyrimidine derivative, and at least one naturally occurring amino acid, or its oligomer or polymer, can be advantageously used to induce and stimulate cell differentiation in the osteogenic and chondrogenic lineage of MSCs. According to the present invention, therefore, it is possible to use the hyaluronic acid derivatives described above for the treatment of skeletal conditions, particularly in the regeneration of bone and cartilage tissues. Still in accordance with the present invention, hyaluronic acid derivatives are advantageously used, as already mentioned, to induce and stimulate cell differentiation in the osteogenic and chondrogenic lineage of MSCs. ML / t / ZUZZ / U / Ί Z40 According to the invention, it is also possible to integrate hyaluronic acid derivatives into suitable pharmaceutical formulations and / or implantable scaffolds, which can be used to support the regeneration of bone and cartilage tissues. The aid derivatives can also be used in therapy for the repair and regeneration of bone and cartilage tissues. An object of the present invention is the use of the derivatives in treatments related to the regeneration of bone and cartilage tissues. According to the present invention, the derivatives can be advantageously integrated into implantable scaffold systems or other pharmaceutically appropriate formulations. Other appropriate pharmaceutical formulations include, but are not limited to, solutions and / or suspensions for parenteral use, solid forms (e.g., tablets, capsules, granules) or semi-solid forms (e.g., gels, pastes, creams, ointments) for oral or topical use, intramuscular and / or subcutaneous implants, and other formulations known to the person skilled in the art. The potential of the invention will now be described in the Experimental Section below, which presents studies carried out using hyaluronic acid derivatives in relation to their potential for cell regeneration of the body's hard tissues. The following examples are provided for illustrative purposes only and are not intended as a limitation. BRIEF DESCRIPTION OF THE FIGURES FIGURES 1A to 1C: Effect of T-Lys on the differentiation of MSCs towards the osteoblastic lineage. Sections: FIGURE 1A: qPCR performed on DBSCs cultured with osteogenic medium and stimulated with 0.3% T-Lys and control DBSCs; FIGURE 1B: Immunoblotting test for the expression of Runx-2 and Col 1 proteins; FIGURE 1C: Histochemical assay on the ALP enzyme (purple staining). FIGURE 2: Effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs. Mineral matrix deposition tested by ARS (red staining) in cells treated with T-Lys, hyaluronic acid and control. FIGURE 3: Effect of T-Lys on the expression of typical markers in chondrocyte cultures. qPCR performed on chondrocyte pellet cultures grown in chondrogenic medium and stimulated with 0.3% T-Lys and a negative control group (Ctr). FIGURES 4A to 4C: Effect of T-Lys on chondrocyte proliferation and tissue growth. Sections: FIGURE 4A: Images and measurements of culture sediments from dissected chondrocytes treated with T-Lys and control groups; FIGURE 4B: Images and measurements of cartilage matrix deposition from dissected chondrocytes treated with T-Lys and control groups; FIGURE 4C: Theoretical reconstruction of the thickness of culture sediments of ML / t / ZUZZ / U / Ί Z40 chondrocytes treated with T-Lys and control groups. DETAILED DESCRIPTION OF THE INVENTION FIGURES 1A to 1C: Effect of T-Lys on the differentiation of MSCs towards the osteoblastic lineage FIGURE 1A: qPCR performed on DBSCs cultured in osteogenic medium and stimulated with 0.3% T-Lys and DBSC Ctr. Each graph represents the mean ± standard error of 3 independent experiments performed in triplicate. *P <0.02 compared to the control group. Expression has been normalized to beta2 microglobulin (B2M). The graphs show that treatment with T-Lys significantly increased the expression of the two osteoblast markers Runx-2 and Col1. FIGURE 1B: Immunoblotting assay for the expression of Runx-2 and Col 1 proteins; each graph represents the calculated average optical density relative to a constituent protein (β-actin maintenance gene) ± standard error from 3 independent experiments performed in triplicate. *P <0.001 compared to the control group. Representative immunoblotting images are also shown on the left side of the figure. The graphs show how the measured parameter is higher in T-Lys-treated cells than in the control group. FIGURE 1C: Histochemical assay of the ALP enzyme (purple staining) performed on DBSCs maintained under osteogenic conditions for 7 days and stimulated with T-Lys compared to the control group. The graph represents the percentage of positive staining relative to the control group (*P < 0.01) and is derived from the analysis of 3 independent experiments performed in quadruplicate. Data are shown as mean ± standard error. Representative images of the culture wells are also shown on the left of the figure. The graph shows how the T-Lys samples have higher alkaline phosphatase enzyme expression. FIGURE 2: Effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs Mineral matrix deposition analyzed by ARS (red staining) in cells treated with T-Lys, hyaluronic acid, and Ctr under osteogenic conditions for 21 days. The graph shows the quantification of the optical density of the dye extracted from the stained cell layers as a mean percentage ± standard error and is representative of 3 independent experiments performed in quadruplicate. *P <0.01, #P <0.001 versus negative control group (Ctr); @P <0.01 versus positive control group (HA). Representative images of the culture wells are also shown on the left of the figure. The graph shows how the T-Lys samples have a higher mineral matrix deposition than both the untreated sample and the sample treated with native hyaluronic acid. FIGURE 3: Effect of T-Lys on the expression of typical markers in chondrocyte cultures. qPCR was performed on chondrocyte sediment cultures grown on chondrogenic medium and stimulated with 0.3% T-Lys and a negative control group (Ctr). Each graph represents the mean ± standard error of 3 independent experiments performed in triplicate. *P <0.04 for Sox-9, *P <0.001 for Col II, *P <0.01 for Col X compared to the control group. Expression was normalized to beta2 microglobulin (B2M). The graphs show that T-Lys treatment significantly increased the expression of the chondrocyte markers Sox-9, Col II, and Col X, while having no effect on Aggrecan expression. FIGURES 4A to 4C: Effect of T-Lys on chondrocyte proliferation and tissue growth FIGURE 4A: Sectioned chondrocyte culture sediments were photographed under a light microscope using a 20x objective lens and analyzed using Image-J software for morphometric examination of the areas. The selected images are representative of three different experiments; the scale bar is shown in the lower right corner of the figures; 75 pm. The graph represents the mean ± standard error of 3 independent experiments performed in triplicate, *P < 0.0003. The sediments treated with T-Lys appear larger than those of the control group. FIGURE 4B: Cartilage matrix deposition was measured using Safranin O staining, and chondrocyte nuclei were counterstained with hematoxylin. Images were taken with a 40x objective lens; scale bar shown in the upper left corner of the figure: 25 pm. The graph represents the mean ± standard error of 3 independent experiments performed in triplicate, *P < 0.04. The number of cells in the T-Lys sample is greater than the number in the control group. FIGURE 4C: The graph shows a theoretical reconstruction of the thickness of the chondrocyte culture sediment in which the number of sections obtained was multiplied by the thickness of the cut and expressed in pm. The group treated with T-Lys shows a greater thickness. EXAMPLES Example 1 Effect of T-Lys on the differentiation of MSCs towards the osteoblastic lineage Dental germ stem cells (DBSCs) were used as a source of MSCs and differentiated for 12 days in osteogenic medium. A portion of the analyzed cells was treated with 0.3% T-Lys (T-Lys treatment group) added to the culture medium with each change. The fraction of cells not treated with T-Lys was used as the control group (Ctr). The mRNA levels of the early markers of typical osteoblasts, Runx-2 and Collagen I (Col 1), were determined in Ctr and T-Lys samples using real-time PCR. ML / t / ZUZZ / U / 1Z40 FIGURE 1A shows how the expression of both markers has increased significantly in T-Lys-treated cells compared to Ctr cells, suggesting that T-Lys treatment has improved the ability of MSCs to differentiate into the osteoblast lineage. The protein expression levels of these osteoblastic markers were further evaluated in T-Lys and Ctr cells by Western blot analysis. FIGURE 1B highlights how the level of Runx-2 and Col 1 proteins increases in T-Lys-treated cells compared to Ctr cells, thus confirming the mRNA expression trend. A histochemical test was then performed to explore the expression of another osteoblastic cell marker, the enzyme alkaline phosphatase (ALP), in response to T-Lys treatment. The result of this experiment, shown in FIGURE 1C, revealed that stimulation of MSCs with T-Lys during osteogenic differentiation significantly increased the purple staining that identifies ALP expression. All the results described above have shown that T-Lys is able to increase the ability of MSCs to differentiate into osteoblast-like cells. Example 2 Effect of T-Lys on mineral matrix deposition during osteogenic differentiation of MSCs In order to thoroughly investigate the effect of this new molecule on the osteogenic differentiation of MSCS, DBSC culture was followed under mineralization conditions for 21 days in different samples: Ctr (without any addition as a negative control group); HA (with the addition of unmodified hyaluronic acid as a positive control group) and T-Lys (in which the cells were treated with 0.3% T-Lys as the treatment group). The effect of T-Lys on DBSC mineral matrix deposition was analyzed using the alizarin red (ARS) staining histochemical test, quantified by a colorimetric technique. The mineralization capacity of cells treated with 0.3% T-Lys proved to be significantly greater than that of both Ctr and HA. These data show how T-Lys is able to increase the osteogenic capacity of MSCs by also stimulating their ability to produce mineralized matrix. Example 3 Effect of T-Lys on the influence of the subcellular distribution of the integrin ανβ3 Integrins are receptors for ECM molecules, important in cell adhesion but also in mediating proliferation and differentiation signals. In particular, the ανβ3 integrin is the receptor for the bone protein osteopontin, which is fundamental in determining the differentiation of MSCs into the lineage. ML / t / ZUZZ / U / 1Z40 osteogenic. Therefore, it was assessed whether treatment with T-Lys could influence the subcellular distribution of the integrin ανβ3. The subcellular distribution of integrin was analyzed using confocal microscopy in DBSCs treated with T-Lys and Ctr cells. Analyses were performed after only 4 days of osteogenic differentiation to compensate for the cells' rapid tendency to form a multilayer, which hinders microscopic observation. In Ctr cells, ανβ3 integrin was found to be distributed across multiple sites, whereas T-Lys treatment induced a different organization of this receptor, more localized to focal adhesion sites. Thus, after 4 days of differentiation, the receptor was still distributed throughout the cell under controlled conditions, while in T-Lys cells it was present only at focal adhesions. The presence of chains (the typical pattern of ανβ3 integrins involved in focal adhesions) was detectable in T-Lys cells but not in Ctr cells.These results suggest that the effect of T-Lys on DBSC differentiation could be mediated by ανβ3 rearrangement. Example 4 Effect of T-Lys on the expression of typical markers in chondrocyte cultures Human joint chondrocytes collected from patients undergoing orthopedic surgery were cultured in pellet cultures to mimic the microarchitecture of three-dimensional tissue and avoid the inappropriate chondrocyte dedifferentiation that readily occurs when cultured in two dimensions. Cell pellets were cultured for 28 days under chondrogenic conditions. The control group (Ctr) was treated according to the conventional protocol, while the T-Lys group was supplemented with 0.3% T-Lys at each vehicle change. At the end of the culture period, the chondrocyte pellet cultures were lysed and evaluated for gene expression analysis. mRNA levels of typical chondrogenic markers—Sox-9, Collagen II (Col II), Collagen X (Col X), and Aggrecan—were determined in both sample groups by real-time PCR. Figure 3 shows the results of these tests, demonstrating that the expression of three of the four markers analyzed was definitely increased in the T-Lys groups. Specifically, the expression of Sox-9, the main transcription factor involved in chondrogenic differentiation, was significantly increased in T-Lys-treated cells compared to Ctr cells. Consistent with this result, Col II and Col X, typical extracellular matrix proteins of cartilage, were also increased by T-Lys treatment, further indicating that this molecule supports and enhances chondrocyte differentiation. On the other hand, the expression of Aggrecan, a cartilage proteoglycan, was unaffected. Example 5 Effect of T-Lys on chondrocyte proliferation and tissue growth After 28 days of differentiation under the conditions described in Example 4, the chondrocyte pellets were fixed with 4% paraformaldehyde, embedded, sectioned, stained, and examined histologically. Morphometric examination by light microscopy of the culture pellets from the dissected chondrocytes revealed that the T-lysates were larger than those in the control group. This result is shown in Figure 4A. To quantify sediment size, samples were sectioned (5 µm thick) and the area of ​​each section, obtained for the two cell groups, was measured using ImageJ software. The graph in FIGURE 4A shows that the average surface area was significantly larger in the T-Lys-treated group than in the control group. The sediments were then stained with Safranin O to highlight the chondrocytes (FIGURE 4B). The staining revealed the presence of a cartilaginous matrix (orange stain), demonstrating that the cells were able to differentiate and produce ECM components under these culture conditions; the nuclei were counterstained with hematoxylin (FIGURE 4B). To verify whether the T-Lys treatment also influenced the cell count, cells were counted in a selected field (100 x 100 µm) for each section. The graph in FIGURE 4B represents the cell count for the two cultures and shows a significant increase in the treated group compared to the control group. Interestingly, a greater number of uniformly thick sections (5 µm), called slices, were obtained from the T-Lys sample compared to the control group. This difference is represented in the graph in FIGURE 4C, where the number of slices has been multiplied by the section thickness (5 pm), thus reconstructing a theoretical thickness of the entire culture sediment. These results demonstrate that TLys stimulates chondrocyte proliferation, differentiation, and matrix secretion. ML / t / ZUZZ / U / Ί Z40 NOVELTY OF THE INVENTION Having described the present invention, it is considered a novelty and, therefore, the contents of the following are claimed as property.

Claims

1. A hyaluronic acid compound and at least one purine and / or pyrimidine-derived heterocycle, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in individual, oligomeric or polymeric form, for use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

2. The compound for use according to claim 1, characterized in that said at least one heterocycle is thymine.

3. The compound for use according to claim 1 or 2, characterized in that said at least one different organic compound is Usina.

4. The compound for use according to any of the preceding claims, characterized in that the bond between said hyaluronic acid, said heterocycle and said naturally occurring amino acid is an ionic chemical bond.

5. The compound for use according to any of the preceding claims, characterized in that said hyaluronic acid compound is T-LysYal®, comprising hyaluronic acid in combination with lysine and thymine.

6. The compound according to any of the preceding claims, for use in the induction and stimulation of cell differentiation in the osteogenic and chondrogenic lineage of mesenchymal stem cells.

7. The compound for use according to claim 6, characterized in that said mesenchymal stem cells are tooth germ stem cells.

8. An implantable scaffold characterized in that it comprises a hyaluronic acid compound and at least one purine and / or pyrimidine-derived heterocycle, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in individual, oligomeric or polymeric form.

9. The implantable scaffold according to claim 8, for use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.

10. A pharmaceutical formulation characterized in that it comprises a hyaluronic acid compound and at least one purine and / or pyrimidine-derived heterocycle, said compound being further associated with at least one different organic compound selected from naturally occurring amino acids in individual, oligomeric or polymeric form, for use in the treatment of skeletal conditions, in particular in the regeneration of hard tissues.